Heterogeneous nuclear ribonucleoprotein U (HNRNPU) safeguards the developing mouse cortex

HNRNPU encodes the heterogeneous nuclear ribonucleoprotein U, which participates in RNA splicing and chromatin organization. Microdeletions in the 1q44 locus encompassing HNRNPU and other genes and point mutations in HNRNPU cause brain disorders, including early-onset seizures and severe intellectual disability. We aimed to understand HNRNPU’s roles in the developing brain. Our work revealed that HNRNPU loss of function leads to rapid cell death of both postmitotic neurons and neural progenitors, with an apparent higher sensitivity of the latter. Further, expression and alternative splicing of multiple genes involved in cell survival, cell motility, and synapse formation are affected following Hnrnpu’s conditional truncation. Finally, we identified pharmaceutical and genetic agents that can partially reverse the loss of cortical structures in Hnrnpu mutated embryonic brains, ameliorate radial neuronal migration defects and rescue cultured neural progenitors’ cell death.

1. The cell death of neural progenitors/stem cells in Hnrnpu fl/fl mutant was not definitely shown. The current data were collected using cultured neurons/slices and using reporter proteins. Since the cortex is still present at E18, although much reduced, the authors should be able to provide data from in vivo studies using apoptosis markers. Alternatively, as the authors were able to perform sgRNA treatment using in utero electroporation (IUE), as shown in Figure 6D, the author should also be able to demonstrate cell death in these embryos. In addition, the number of neural progenitors needs to be compared in vivo at different stages (such as by EdU labeling) to explain the progressive loss of stem cells. Similarly, the rescue of the progenitor loss by deleting Tp53 in the Hnrnpu fl/fl background needs to be demonstrated using the abovementioned in vivo assays. The reviewer understands this is significant amount of work, but feels that it is necessary to support the claim that Hnrnpu is required for the survival of neural stem cells, but not for postmitotic neurons. Figure 5C, activated caspase 3 staining was widespread and was not restricted to the VZ/SVZ, arguing against the notion that Hnrnpu truncation primarily affects neural progenitor survival. By contrast, in Figure 6D, in utero electroporation of Hnrnpu sgRNA did not appear to cause significant thinning of the cerebral wall due to the loss of neural progenitors, although the migration of upper layer neurons was visibly reduced. Therefore, it is not clear from the existing data where cell death occurs.

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3. It is not specified if the sgRNA treatment against Hnrnpu generates the same truncated HNRNPU or eliminates the protein completely. These two genetic conditions are most likely to affect the gene function differentially, as the truncated protein is expected to only disrupt the RNA-binding ability. Along the same line, the authors suggested that the amount of the truncated protein is reduced in the floxed allele in Figure 5. However, western blotting in supp Figure 1F showed that the levels of truncated and full-length proteins were comparable. Therefore, the genetic conditions resulting from Cre-mediated and sgRNA-mediated deletions are not necessarily the same, or even similar.
4. The effects of Hnrnpu truncation/deletion on Mdm2 are not consistent among the figures. For example, Figure 4C showed a reduction in the overall level of Mdm2. This was not the case in Figure  3E or Figure 6C. In addition, the alternative splicing patterns for Mdm2 were different between Figure Figure 6C & D, Srsf3 and Hnrnpu sgRNA treatment appeared to cause similar effects in Mdm2 alternative splicing (i.e., exon 3 was more skipped; Srsf3 had a milder effect) and in reducing neuronal migration (Srsf3 had a stronger effect here). This contradicts the notion that the two splicing factors have opposing effects.

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6. In all figures demonstrating the cortex (other than Figure 6D), there was no labeling of the orientation or of the layers, making it very difficult to assess the phenotypes. 7. In Figure 1A, why was EdU seen in superficial layers after 1 hr labeling? Taken together, in vivo studies using the floxed mutant and/or IUE, and detailed temporal and spatial analyses of apoptosis are much needed. They will help demonstrate where cell death occurs in vivo as a result of Hnrnpu deletion or truncation.
Minor issues: 1. WT1 and WT2 in Figure 5 and Supplementary Figure 5 should be labeled as Control1/Ctrl1 and Control2/Ctrl2, as the animals contained transgenic alleles. Figure 4 is too low to view.

The resolution of Supplementary
3. fl/fl was used to indicate the deletion of Hnrnpu, but loxP/loxP was used for Tp53. It'd be better to be consistent. Figure 5 was titled "Rescue of alternative splicing of MDM2 and VZ organization in Hnrnpu mutant cortices following Tp53 KO". However, Tp53 KO does not alter the AS by itself and also does not restore the AS in Hnrnpu fl/fl background. The title is therefore misleading. "E24 WT cortices" in the same legend should be E14. Figure 1F-I, sections were labeled as 5 µM. Should be 50 µm?

REVIEWER COMMENTS
Reviewer #1 (Remarks to the Author): Sapir et al. reported in this manuscript that the HNRNPU protein is required for embryonic mouse brain neuronal progenitors to survive. The expression and alternative splicing of multiple genes involved in cell survival, cell motility, and synapse formation are affected following Hnrnpu's conditional truncation. They also identified pharmaceutical and genetic agents that can rescue neuronal stem cell death were able to restore neuronal migration. They conclude that their studies point to novel roles of HNRNPU during brain development. In general, the study was well conducted, and the observations are interesting and potentially important for better understanding the roles of HNRNPU in brain development. However, there is a major concern that needs to be addressed before reaching a clear and solid conclusion.
Major Concern: 1) The authors used Emx1Cre mice to delete/truncate HNRNPU in neuronal stem cells and neurons. However, Emx1 is also expressed in astrocytes and oligodendrocytes, leading to HNRNPU deletion/truncation also in astrocytes and oligodendrocytes. The authors should experimentally test the contributions of potential astrocyte and oligodendrocyte deficits to the observed brain development deficits.
We thank the reviewer for raising the issue of non-neuronal cells. To address this issue, we performed several experiments, which we present hereafter. Our observations suggest that the death of Emx1::Cre expressing cells that carry two copies of Hnrnpu floxed alleles and their progeny is progressive and extensive. We observed that the cell death starts medially and progresses laterally, in a gradient that mirrors the expression pattern of Emx1. We first looked at E18 brains from controls, Hnrnpu +/+ Emx1 Cre/+ , and Hnrnpu fl/fl Emx1 Cre/+ littermates. We stained these brains with CM5 (anti-Tp53, Leica) to map the region where p53 is stabilized (Fig. 4 C-F). We used glial fibrillary acidic protein (GFAP) to stain astrocytes that occupy the cortical plate ( Fig. 1 J-P). We additionally immunostained the sections using anti-TBR1 antibodies ( Fig.1 K, N ). TBR1 is expressed soon after cortical progenitors differentiate and is highly expressed in early-born neurons that occupy the deep cortical layer 6. We noticed that at E18, the medial parts of the cortex of Hnrnpu fl/fl Emx1 Cre/+ embryos were lost, and the lateral ventricle seemed to be exposed (Fig. 1J, Fig. 4E). This region is rich in cells with high levels of nuclear TP53. Since more laterally, a structure resembling the cortex is still visible, we determined that the superficial regions of the remaining cortical structure are GFAP positive, yet markedly reduced levels of GFAP staining are detected. The deep layer 6, TBR1 positive cells no longer resided at a deep position but are spread throughout the width of the cortex ( Fig.1 K vs. N). To estimate the reduction in GFAP signal and distribution, we produced intensity histograms from the upper part of the cortex in sections of n=3 brains for each genotype (Fig. 1 P). Both genotypes showed a signal peaking in the basal edge of the cortical plate, yet they were remarkedly different. We found that the integration of the signal over 300 microns (area under the curve) was significantly reduced in the mutant compared to control brains (13825±209.1 units in the control sections vs. 8461± 168.4 in the mutant brains, n=3, unpaired t-test, P<0.0001).
In addition, we looked at P21 brains. This time point was the most advanced age in which mutants could be collected as they could not survive after weaning. The entire cortical region is missing at this age, as seen as early as P8 (Fig.  1I, schematically shown next to panel T). P21 brain sections of mutant and control (Hnrnpu +/+ Emx1 Cre/+ ) littermates were stained with deep and superficial cortical plate layers markers (Tbr1, Cux1 Fig 1Q, T) 2′,3′-cyclic nucleotide-3′phosphodiesterase (CNPase) that stains myelinating glia and myelin (Fig. 1R, U) and GFAP ( Fig. 1 S, V). Although we could detect at the appendix that persists at the most ventrolateral region of the cortex both GFAP and CNPase and some positive neuronal markers, the area lacks any organization. It could not be assigned an anatomical annotation. Due to this dramatic phenotype, we chose a subtler approach (Supp. Fig 1). We lineage traced (E14 to P21) cells treated with either Hnnrpu sgRNA or control px330 plasmid. To this end, we electroporated E14 embryos with a combination of CAG::GFP and transposase-transposon system containing pCAG::PBASE and a dsRed donor plasmid, pB CAG::DsRed. This experiment allowed us to distinguish lineage traced astrocytes and earlier born neurons (Supp. Fig. 1A-G). The first-born neurons expressed GFP from the episomal plasmid CAG::GFP and dsRed from the piggybac one and resided in layers II-IV. The later-born progeny appeared red as the cells lost the episomal GFP but expressed dsRed from the integrated transposon. These cells exhibited an astrocytic appearance and expressed GFP under a GFAP promoter (data not shown). We used CRISPR/CAS9 constructs targeting the Hnrnpu locus as an effective way to reduce HNRNPU levels (see Supp. Fig. 8). We sacrificed the mice (n=3 of each treatment at P21) and calculated the proportion of the dsRed+GFP-out of all labeled cells (single or double-labeled, Supp. Fig 1 H -J). We noticed that the percentage of late-born astrocytes at P21 was similar, regardless of HNRNPU levels. Some ectopic neurons could be detected in the Hnrnpu sgRNA's treated brains consistent with migration deficits that we reported in the main text. Taken together, we concluded that the progenitor's death affected all their progeny, neurons and astrocytes alike, but could not evaluate the relative vulnerability of astrocytes relative to neurons as a result of lower dosage of HNRNPU. The new results are incorporated to Fig 1, Fig 4, and Supp. Fig 1. We addressed this point in the modified discussion.
Neural progenitors were highly vulnerable to HNRNPU protein depletion, whereas postmitotic neurons in vitro and neurons and astrocytes in vivo did not display similar sensitivity to HRNPU loss. This difference was not correlated with the expression pattern since HNRNPU was highly expressed in radial glia as well as in postmitotic neurons and astrocytes.
Minor Comments: 1) The authors seemed inter-changeably used neural stem cell versus neuronal stem cell in the manuscript. Since they are different, the authors should be consistent and define clearly what they exactly mean. Following this remark, the term "Neuronal" was changed to "neural," and the term "Stem cells" was changed to "progenitors".
This study was done in the earliest time point of E13-E14 mouse brains, populated by neural progenitor cells (NPCs), the progeny of neuroepithelium division. NPCs either self-proliferate or give rise to intermediate progenitors and neurons and later glial lineages. The text was changed accordingly.
2) All n numbers for each experiment should be clearly provided in each figure legend.
Numbers were added wherever they were missing.

Reviewer #2 (Remarks to the Author):
This study by Sapir, Reiner, and colleagues examined the function of the Hnrnpu gene, whose dysregulation is associated with various human disorders. The authors focused on the developmental role of the gene in the cortex and used genetic, molecular, and imaging approaches to address the question. The authors identified the p53 pathway, amongst others, as key molecules affected by HNRNPU truncation. The authors also achieved a partial rescue of HNRNPU truncation by deleting Tp53 or by using various cell death inhibitors. Although the subject is interesting and significant, the evidence presented here has major weaknesses as outline below, which renders the conclusion unconvincing.
Major issues: 1. The cell death of neural progenitors/stem cells in the Hnrnpu fl/fl mutant was not definitely shown. The current data were collected using cultured neurons/slices and using reporter proteins. Since the cortex is still present at E18, although much reduced, the authors should be able to provide data from in vivo studies using apoptosis markers.
Alternatively, as the authors were able to perform sgRNA treatment using in utero electroporation (IUE), as shown in Figure 6D, the author should also be able to demonstrate cell death in these embryos. In addition, the number of neural progenitors needs to be compared in vivo at different stages (such as by EdU labeling) to explain the progressive loss of stem cells. Similarly, the rescue of the progenitor loss by deleting Tp53 in the Hnrnpu fl/fl background needs to be demonstrated using the abovementioned in vivo assays. The reviewer understands this is significant amount of work, but feels that it is necessary to support the claim that Hnrnpu is required for the survival of neural stem cells, but not for postmitotic neurons.
We thank the reviewer for these insightful comments. We indeed followed the suggestion to look at the persistent cortex at E18. We compared E18 brains from controls, Hnrnpu +/+ Emx1 Cre/+ , and Hnrnpu fl/fl Emx1 Cre/+ littermates immunostained these brains with CM5 (anti-Tp53, Leica) to map the region where p53 is stabilized (Fig 4 C-F). We could detect Tp53 accumulation in cells throughout the entire width of the remaining cortex. We could not use this data to support or dispute the notion that death occurs in a subset of cells.
Alternatively, as the authors were able to perform sgRNA treatment using in utero electroporation (IUE), as shown in Figure 6D, the author should also be able to demonstrate cell death in these embryos.
Based on our in vitro data (Fig. 2), we concluded that CAG-driven Cre deletion of Hnrnpu floxed alleles causes progenitors' death. At the same time, late activation of Cre (under T promoter) allows some neurons to survive. Similarly, we found that using in utero electroporation, the introduction of Hnrnpu sgRNA at E14 enables neurons to persist with reduced levels of HNRNPU (Supp. Fig 8). To test the extent of cell death in brains treated with Hnrnpu sgRNA, we followed the reviewer's suggestion and electroporated brains with either Hnrnpu sgRNAs, Srsf3 sgRNAs, a combination of both, or control px330. The brains were collected four days post EP, and the sections were immunostained with Cleaved Caspase 3 (CC3). The findings are presented in Supp. Fig. 9, showing sporadic cells that appear to be CC3 positive in both the Hnrnpu sgRNAs and Srsf3 sgRNAs and none in which both Hnrnpu and Srsf3 were knocked out. Yet, due to the small number of cells, we may conclude that in utero, cell death that is seen four days after in utero introduction of CRISPR/CAS9 Hnrnpu sgRNA is minimal, and no strong statements can be made regarding the effect of the different sgRNA treatments.
These new results are shown in Supp. Fig. 9.
In addition, the number of neural progenitors needs to be compared in vivo at different stages (such as by EdU labeling) to explain the progressive loss of stem cells.
Based on our data and the E14-18 experiment presented earlier, we claimed that progenitors present higher sensitivity to Hnrnpu loss of function. We injected EdU to E14 pregnant mice 30 minutes before sacrificing the female to look at their progressive loss. We immunostained the brain sections with anti-Sox2 antibodies that mark selfrenewing neural stem cells and anti-Tbr2 antibodies that mark intermediate progenitors. We focused on the regions immediately adjacent to the extensive cell death at the medial part of the Emx1 expressing domains. We found that mutant brains' ventricular/subventricular zones (VZ/sVZ) express Sox2 and Tbr2. These areas show a dramatic size reduction and display a marked decrease in EdU incorporation (Supp. Fig. 3). EdU incorporation reduction was measured and presented later in the manuscript (Fig. 5AE); see next paragraph.
These results are now presented in Supp. Similarly, the rescue of the progenitor loss by deleting Tp53 in the Hnrnpu fl/fl background needs to be demonstrated using the abovementioned in vivo assays. The reviewer understands this is significant amount of work, but feels that it is necessary to support the claim that Hnrnpu is required for the survival of neural stem cells, but not for postmitotic neurons.
We followed the reviewer's advice and have used EdU to label S phase cells in the cortical plate of E14 littermates. We obtained three rescued brains (Hnrnpu fl/fl Emx1 Cre/+ Tp53 loxP/loxP ). In those mice, we observed rescue of the cortical structure and could count the number of cells in identical areas (100x100 M 2 ) the sVZ of coronal sections (14M thick) obtained from these brains and three control brains. Control brains were analyzed for the same litters as the rescued brains. We found that the number of EdU cells in the rescued brains was reduced to 31.7%±3.07 of control values (Average ± SEM, of n=3 brain, 23 data points). These were partially restored to 56.97% ±2.65 compared to the control in the rescued brains (Fig. 5AE). We find this number consistent with our estimation of a partial rescue of the cortices by avoiding p35-dependent cell death.
EdU quantification was included in Fig. 5 AE. Figure 5C, activated caspase 3 staining was widespread and was not restricted to the VZ/SVZ, arguing against the notion that Hnrnpu truncation primarily affects neural progenitor survival. By contrast, in Figure 6D, in utero electroporation of Hnrnpu sgRNA did not appear to cause significant thinning of the cerebral wall due to the loss of neural progenitors, although the migration of upper layer neurons was visibly reduced. Therefore, it is not clear from the existing data where cell death occurs.

In
We thank the reviewer for this observation. We indeed saw widespread CC3 staining in E14 Hnrnpu fl/fl Emx1 Cre/+ brain sections (Fig. 5 C). We continued and confirmed that p53 accumulation appears in cells in the entire width of the dying cortex (Fig. 4 C-F). Additionally, we stained electroporated brains with CC3 and observed merely sporadic cell death four days post IUE (Supp. Fig. 9). The electroporated brains are, in fact, a mosaic of electroporated cells in a wild-type environment. The reduction in HNRNPU levels in the cells is impressive, but there is variability in the electroporated cells (See Supp. Fig. 8). Additionally, the introduction of the CRISPR/CAS9 Hnrnpu sgRNA in utero is done at E14, and there is some delay until the construct is expressed (we estimate at least 12 h, based on our in vitro studies (Fig. 4B), the gene is deleted, and the reduction in HNRNPU levels occurs. These differences between the two experimental systems make it hard to conclude whether the loss of HNRNPU results in a specific cell-type sensitivity. We used E14 slices from EdU treated control and mutant embryonic brains to map the progression of cortical tissue loss (Supp. Fig 3). We indeed saw a massive reduction in the VZ/sVZ (Sox2, Tbr2 positive cells). The cortical plate regions are missing in medial areas and sparse more ventrally. Since the CP is the progeny of the VZ/sVZ, this can be a secondary effect. We did observe some early-born layer six neurons in the cortical structures that still exist at E18 but are almost entirely gone at P8 and P21 (Fig 1 H-I and Q-U). We, therefore, believe that the question of which cells are more sensitive to Hnrnpu loss-of-function is not conclusive based on the analysis of Hnrnpu deleted brains. We suggest that the experiments presented in Figure 2 and Supplementary Fig. 2 are more informative and support the notion of progenitors' death and more considerable sensitivity to HNRNPU loss of function.
3. It is not specified if the sgRNA treatment against Hnrnpu generates the same truncated HNRNPU or eliminates the protein completely. These two genetic conditions are most likely to affect the gene function differentially, as the truncated protein is expected to only disrupt the RNA-binding ability. Along the same line, the authors suggested that the amount of the truncated protein is reduced in the floxed allele in Figure 5. However, western blotting in supp Figure 1F showed that the levels of truncated and full-length proteins were comparable. Therefore, the genetic conditions resulting from Cre-mediated and sgRNA-mediated deletions are not necessarily the same, or even similar.
We agree that both treatments are not identical. Based on others that used the same truncated allele (Ye et al., 2015) (Bagchi et al., 2020) we would like to suggest that the deletion of Hnrnpu causes loss of function. Moreover, we observed a reduction in anti-HNRNPU reactivity by immunohistochemistry in brains in which the HNRNPU is truncated when we used an antibody that was raised against the N terminus of the protein, suggesting that the truncated product is either present in reduced levels or is unstable (See Figure 5

S vs. U)
We would like to present these images to the reviewer showing slices from E14 control (A) mutant (B) and rescued (C) brains, stained with anti-N-ter HNRNPU, that reacts with the full length and the truncated HNRNPU protein (yellow). Most cells in the mutant (B) and rescue (C) cortex do not express HNRNPU, suggesting that the truncated protein is not stable. The cells that are HNRNPU+ scattered in the rescue and mutant cortices are likely interneurons that are invading the cortex from the HNRNPU+ ganglionic eminence that do not express Emx1. Bones and meninges are still visible (and contain HNRNPU+ nuclei) as sections were done on the embryo head without prior removal of the skull.
4. The effects of Hnrnpu truncation/deletion on Mdm2 are not consistent among the figures. For example, Figure 4C showed a reduction in the overall level of Mdm2. This was not the case in Figure 3E or Figure 6C. In addition, the alternative splicing patterns for Mdm2 were different between Figure 3E  Total RNA seq (done on mice cortices) provided quantitative data on both expression levels of all splice variants and highlighted dysregulation of splicing events in intronic levels and gene levels. To verify the under/over-representation of specific splice variants, we performed cDNA using splice variant-specific primers. Both experiments show the difference in the representation of the targeted splice variance, yet the PCR shown in Fig. 3E, Fig 6C, and Supp. Fig.  7A is not suitable to measure expression levels. To obtain quantitative data, we co-electroporated cultured progenitors (Neutrospheres, NS) with pCAG::GFP and CRISPR/CAS9 targeting Hnrnpu, Srsf3 sgRNA, or both, and compared the data to control NS electroporated with pX330. We performed qPCR on cDNA prepared from each treatment and measured the relative levels of Mdm2 splice variants (MDM2 del E3/MDM2). The results are included in Fig. 6D. Figure 6C & D, Srsf3 and Hnrnpu sgRNA treatment appeared to cause similar effects in Mdm2 alternative splicing (i.e., exon 3 was more skipped; Srsf3 had a milder effect) and in reducing neuronal migration (Srsf3 had a stronger effect here). This contradicts the notion that the two splicing factors have opposing effects.

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We cited that the general accepted notion is that " SR (serine/arginine-rich) splicing factors (SRSF) generally oppose HNRPs with exon skipping and exon retention (Busch and Hertel, 2012;Van Nostrand et al., 2020)." However, we agree with the reviewer that in each individual splicing event, the effects will differ. It should be noted that both HNRNPU and SRSF3 are members of large protein families that may exhibit, in some cases, redundant and overlapping activities. More specifically, in the experiments we show, the simultaneous manipulation of both genes results in a functional improvement. We modified the text accordingly.
6. In all figures demonstrating the cortex (other than Figure 6D), there was no labeling of the orientation or of the layers, making it very difficult to assess the phenotypes.
We appreciate this remark as the mutant brains are so disturbed their phenotype may be hard to assess. We made an effort to orient the readers better by adding low magnification images of a vast area of the brain and annotating major structures (Fig. 1B We also reoriented the mutant brains in a dorsal up ventral down orientation and indicated the imaged regions, hoping that this will clarify the data. 7. In Figure 1A, why was EdU seen in superficial layers after 1 hr labeling? The following publications presented pulse labeling with Thymidine Analogues such as BrdU of E14.5 mouse embryos. Both shorter pulses (30 min) or similar pulse to the one the reviewer raised concern, (1h) show superficial cells that are BrdU positive Panels C, E (Pucilowska et al., 2012), and Panel D (Hou et al., 2012). These cycling cells may belong to the meninges, and they are not included in our analysis (we only refer to cells in the VZ and sVZ of the labeled brains) https://www.jneurosci.org/content/32/25/8663.long (Pucilowska et al., 2012)

Figure 5.
Loss of ERK2 disrupts basal progenitor frequency and generation, resulting in premature progenitor pool depletion. b, the number of intermediate progenitors was analyzed by immunohistochemical analysis with Tbr2 (red) at E14.5 (p = 0.0080). A short BrdU (50 ng/g) pulse was intraperitoneally injected 30 min before sacrificing the pregnant dam. c, d, The number of BrdU+ cells (green) was counted at E14.5. e, f, To analyze the frequency of cycling SVZ progenitors, we used a short-pulse BrdU labeling paradigm (green) to immunolabel Tbr2+ (red) progenitors in the S phase, Tbr2+/BrdU+ (n = 5; p = 0.0022). g, h, Basal progenitor generation from apical progenitors was assayed by co-labeling Tbr2+ cells with BrdU 16 h post-BrdU injection, allowing some apical progenitors to migrate into the SVZ and express Tbr2 (red) (n = 5; p = 0.0001).
Taken together, in vivo studies using the floxed mutant and/or IUE, and detailed temporal and spatial analyses of apoptosis are much needed. They will help demonstrate where cell death occurs in vivo as a result of Hnrnpu deletion or truncation.
Minor issues: 1. WT1 and WT2 in Figure 5 and Supplementary Figure 5 should be labeled as Control1/Ctrl1 and Control2/Ctrl2, as the animals contained transgenic alleles. We apologize for the low-quality image; it is now replaced with a new figure (Supp. Fig. 6).
3. fl/fl was used to indicate the deletion of Hnrnpu, but loxP/loxP was used for Tp53. It'd be better to be consistent.
The nomenclature we use follows the nomenclature of the original papers that created these mice lines with minor modifications. (Ye et al., 2015) describe the line as: "control (Hnrnpu f/f )". We used the abbreviation fl for floxed. (Marino et al., 2000) refer to the conditional p53 mice as "p53 LoxP/LoxP" Here, we used the gene name Tp53 but left the loxP nomenclature as is. As several labs shared both these lines, we felt that this would be easier for the scientific community to place them in the context of the previously published manuscripts. Figure 5 was titled "Rescue of alternative splicing of MDM2 and VZ organization in Hnrnpu mutant cortices following Tp53 KO". However, Tp53 KO does not alter the AS by itself and also does not restore the AS in Hnrnpu fl/fl background.

Supplementary
We thank the reviewer for noting this. Indeed, Tp53 KO prevents cell death but does not alter the splicing of MDM2. The title of the figure (Now Supp. Fig. 7) was corrected accordingly.
The title is therefore misleading. "E24 WT cortices" in the same legend should be E14.
The labels in the Figures are correct. The size of the bar in panel A' is approximately the size of one cell nucleus. The size of the bars in G and I are in mm.